Structural basis of antagonist selectivity in endothelin receptors

Endothelins and their receptors, ETA and ETB, play vital roles in maintaining vascular homeostasis. Therapeutically targeting endothelin receptors, particularly through ETA antagonists, has shown efficacy in treating pulmonary arterial hypertension (PAH) and other cardiovascular- and renal-related diseases. Here we present cryo-electron microscopy structures of ETA in complex with two PAH drugs, macitentan and ambrisentan, along with zibotentan, a selective ETA antagonist, respectively. Notably, a specialized anti-ETA antibody facilitated the structural elucidation. These structures, together with the active-state structures of ET-1-bound ETA and ETB, and the agonist BQ3020-bound ETB, in complex with Gq, unveil the molecular basis of agonist/antagonist binding modes in endothelin receptors. Key residues that confer antagonist selectivity to endothelin receptors were identified along with the activation mechanism of ETA. Furthermore, our results suggest that ECL2 in ETA can serve as an epitope for antibody-mediated receptor antagonism. Collectively, these insights establish a robust theoretical framework for the rational design of small-molecule drugs and antibodies with selective activity against endothelin receptors.


INTRODUCTION
Endothelins are pivotal regulators of cardiovascular functions, essential for maintaining vascular tone and overall cardiovascular homeostasis 1 .Three endothelin peptides, namely ET-1, ET-2, and ET-3, are characterized by two unique cysteine-cysteine crosslinks 2 and activate the endothelin receptor (ETR) subtypes ET A and ET B 3,4 .Notably, human ET A and ET B receptors share 63% sequence homology 3 but differ significantly in their ligand affinity and function.For instance, ET A preferentially binds ET-1 and ET-2 over ET-3 5 , mediating strong vasoconstriction, whereas ET B exhibits equal affinity for all three isoforms, primarily inducing vasorelaxation through nitric oxide and facilitating ET-1 clearance 6,7 .Consequently, ETRs are crucial targets for the treatment of cardiovascular diseases 8 .
Given the complex role of ETRs, the development of therapeutic drugs has primarily focused on vasodilatory antagonists for treating pulmonary arterial hypertension (PAH) and autoimmune diseases 9,10 .Notable selective ET A antagonists or dual ET A /ET B antagonists include bosentan, macitentan, and ambrisentan [11][12][13][14][15] .Additionally, ETR antagonists like sparsentan, aprocitentan, and zibotentan are currently under investigation for their potential efficacy in treating refractory hypertension and various kidney diseases [16][17][18][19] .Conversely, ET B agonists are being explored for therapeutic benefits such as vasodilation and neuroprotection 20,21 .Furthermore, the development of therapeutic vaccines and monoclonal antibodies targeting ET A represents an exciting frontier in PAH treatment, combining high specificity with a reduced risk of side effects 22 .Preclinical studies have shown promise in these approaches to decrease pulmonary arterial pressure 23,24 .
Structural studies on ETRs have elucidated the mechanisms of endothelin ligand recognition and activation.Extensive X-ray crystallography work has revealed the interaction patterns within the ET B receptor when bound with various ligands, including ET-1, ET-3, and several antagonists 5,[25][26][27][28][29] .Complementarily, recent cryoelectron microscopy (cryo-EM) studies on ET A and ET B in complex with G protein in active states have provided insights into the conserved recognition mechanisms for endogenous agonists and the selectivity for synthetic agonists between ET A and ET B 30 .Despite these advances, the structural basis for ET A antagonism remains less understood, a gap that limits the design of selective antagonists.In addition to small-molecule antagonists, a monoclonal antibody (Fab 301 ) specifically targeting ET A is currently in phase Ib clinical trials 23 .The specificity of antibodies may address the selectivity issues associated with small-molecule antagonists for ETRs 22,24 .However, there is currently no structural information on antibody-receptor complexes 24,31 .
This study aims to address these questions by presenting cryo-EM structures of human ET A in complex with key small-molecule antagonists and Fab 301 and by detailing the activation mechanisms of both ET A and ET B receptors.These molecular insights are crucial for advancing the development of therapeutics for conditions such as PAH and are part of broader efforts to enhance the specificity and efficacy of ETR-targeted drugs.

Structure determination of antagonist-bound ET A structures
To understand the structure basis for the antagonist binding modes in ET A , we analyzed three distinct compoundsmacitentan, ambrisentan and zibotentanmeasuring their activities on ET A in calcium mobilization assay (Fig. 1a; Supplementary Fig. S1).Determining the structure of inactive-state G protein-coupled receptors (GPCRs) via cryo-EM is inherently challenging due to the absence of the heterotrimeric G protein, which is essential for particle alignment in active GPCR structure determination.Here, we developed an optimized strategy to solve the inactive ET A structures.Initially, to overcome the low surface expression of ET A , endoglucanase H was fused to the N-terminus of ET A , along with N-and C-terminal truncations (Materials and methods).Then, to compensate for the absence of the G protein, a thermostabilized apocytochrome b562 RIL (BRIL) 32 protein was fused between TM5 and TM6, replacing ET A 's third intracellular loop (ICL3) (Supplementary Fig. S1a).The design of the BRIL fusion sites for uninterrupted helicity was informed by AlphaFold2 33 predictions.
Subsequent stabilization of the modified ET A construct involved the introduction of a BRIL-binding Fab and a nanobody (Nb) that reinforces the hinge region, as previously described 34,35 .Despite these modifications, we encountered stability and orientation preferences during cryo-EM grid preparation.To address these issues, we introduced an ET A -specific antibody, Fab 301 23 , which not only conferred additional stability to the complex but also exhibited antagonistic effects against ET-1-induced ET A signaling.These strategic modifications enabled us to determine the cryo-EM structures of Fab 301 -bound ET A in complex with macitentan, ambrisentan and zibotentan, at nominal resolutions of 3.1 Å, 3.2 Å and 3.2 Å, respectively (Fig. 1b−e; Supplementary Fig. S2 and Table S1).The high-resolution cryo-EM maps provided detailed electron density in the orthosteric ligand-binding pocket, enabling accurate placement of each antagonist (Supplementary Fig. S2).It is noteworthy that the interaction interface between Fab 301 and ET A is quite limited, and within the cryo-EM particles, Fab 301 appears to exhibit a degree of movement.
Comparative analysis of the macitentan-ET A , ambrisentan-ET A and zibotentan-ET A complexes reveals a conserved conformation among the three structures, with C α root mean square deviation (RMSD) values between 0.7 Å and 1.0 Å (Supplementary Fig. S3a,  b).Compared to the crystal structures of antagonist-bound ET B , both receptors adopt a similar inactive conformation, with an RMSD of 1.5 Å (Supplementary Fig. S3c).

Binding of macitentan to ET A
Macitentan, designed as an ET A -selective antagonist, is a derivative of the dual-acting antagonist bosentan.In our calcium mobilization assay, macitentan shows a high affinity to ET A , with an IC 50 of 1.3 nM (Fig. 1a; Supplementary Table S2), contrasting to the lower affinity to ET B at 14.5 μM (Supplementary Fig. S4b), which is also consistent with the previous research results 36 .Structurally, macitentan retains the pyrimidine core characteristic of bosentan but distinguishes itself with a sulfonamide substitution at the fourth position 37 (Supplementary Fig. S1b, c).The binding of macitentan within the ET A pocket involves several distinct interactions (Fig. 2a).The sulfonamide moiety in macitentan establishes a hydrogen bond with R326 6.55 (Fig. 2b), and forms ionic interactions with residues K166 3.33 , K255 5.38 and R326 6.55 from ET A (Fig. 2b).The bromophenyl group is embedded in the receptor's hydrophobic core, forming hydrophobic interactions with residues W319 6.48 , V169 3.36 , Y263 5.46 , H323 6.52 , and L259 5.42 from TMs 3, 5, and 6 (Fig. 2c), and is further stabilized by a cation-π interaction with K166 3.33 (Fig. 2b).The 2-(5-bromopyrimidin-2-yl)oxyethoxy component, linked to the sixth position of the pyrimidine, allows the oxygen atom in the bromopyrimidine to form a hydrogen bond with Q165 3.32 (Fig. 2d).Importantly, this moiety inserts deeper into the orthosteric pocket than the similar groups in bosentan or K-8794 in ET B , positioned to form a halogen bond with D126 2.50 (Fig. 2d).Furthermore, bromopyrimidine engages in π-π interactions with Y129 2.53 and the "toggle switch" residue W319 6.48 (Fig. 2d).The ethyl tail attached to the sulfonamide moiety is nestled within a hydrophobic pocket formed by F161 3.28 , P162 3.29 , and F224 4.64 from TM3 and TM4 of ET A .Prior research has demonstrated that substituting the sulfonamide moiety with sulfamide significantly boosts ETR antagonists' receptor affinity 11 .Our structural analysis reveals that the addition of the -NH-group in macitentan contributes to a more stable electrostatic network with K166 3.33 and K255 5.38 , enhancing macitentan's binding affinity (Fig. 2b).Notably, the R326 6.55 A mutation showed minimal influence on macitentan's activity on ET A , aligning with macitentan's stronger electrostatic network at this location in ET A (Fig. 2m).
Binding of ambrisentan to ET A Ambrisentan, a propionic acid derivative discovered through highthroughput screening, differentiates itself from macitentan by featuring a carboxylic acid group instead of a sulfonamide moiety 15 (Supplementary Fig. S1d).Despite its smaller molecular weight relative to macitentan and zibotentan, ambrisentan exhibits a remarkably high affinity to ET A , befitting its role as an ET A -selective antagonist (Fig. 1a).The structural elucidation of its binding to ET A uncovers essential interactions.The carboxylic acid group forms ionic interactions with the positively charged residues K166 3.33 and R326 6.55 (Fig. 2f), while a network of hydrogen bonds with residues R326 6.55 , K166 3.33 , and Q165 3.32 firmly anchors ambrisentan in the binding site (Fig. 2e, f).Moreover, the hydrophobic interaction of one benzene ring from the symmetric pair in ambrisentan with residues F161 3.28 , P162 3.29 , and F224 4.64 creates a snug fit within a subpocket (Fig. 2f).The Fig. 2 Characterization of macitentan, ambrisentan, and zibotentan binding modes in ET A .a-d Schematic of macitentan's interactions with key residues in ET A .e-h Schematic of ambrisentan's interactions with key residues in ET A .i-l Schematic of zibotentan's interactions with key residues in ET A .The selectivity of zibotentan towards ET A may be attributed to Y129 2.53 in ET A but H150 2.53 in ET B (l). m-o The antagonistic effects of four different antagonists on the ET A R326 6.55 A variant (m); effects of mutations in ET A (n) and ET B (o) on zibotentan's antagonistic activity.ΔpIC 50 represents the difference in pIC 50 values between the wild type (WT) and the mutants of ET A .Data are presented as means ± SEM (n ≥ 3).Hydrogen bonds are highlighted with gray dashed lines.
Subsequent structural investigations suggest that zibotentan's selectivity for ET A may be significantly influenced by the residue F161 3.28 , which is a valine (V177 3.28 ) in ET B .This phenylalanine F161 3.28 acts as a "tray" that stabilizes one end of the zibotentan molecule (Fig. 2l).Concordantly, mutation of F161 3.28 A or F161 3.28 V significantly reduces zibotentan's activity on ET A (Fig. 2n).Additionally, the interaction with Y129 2.53 is also crucial; its alteration to phenylalanine or histidine (Y129 2.53 F or Y129 2.53 H) markedly reduces zibotentan's activity on ET A , highlighting the importance of this residue for antagonist specificity (Supplementary Fig. S4c−e).Intriguingly, introducing corresponding mutations into ET B (V177 3.28 F and H150 2.53 Y) partially impairs the activity of zibotentan on ET B , which underscores the critical role of these residues in the determination of antagonist selectivity in ET A or ET B subtype (Fig. 2o).

Structure basis of antagonist selectivity in ET A and ET B
Structural elucidation of ET A in complex with various antagonists, each featuring distinct scaffold architectures, advances our understanding of the antagonist binding modes in ET A and delineates commonalities critical for their antagonistic function.An important aspect of their activity is the interaction with the positively charged region in ET A 's orthosteric pocket.Functionally important groups, such as the sulfonylamide in bosentan, macitentan, and zibotentan, along with the carboxylic acid group in ambrisentan, are essential.These functional groups occupy the position corresponding to W21, the C-terminal end of the endogenous agonist ET-1 (Fig. 3a), a key engagement site with ET A 30 .Another shared trait of these antagonists is their interaction in close proximity to TM5 and TM6, which helps stabilize ET A conformation in this region (Fig. 3b).The three antagonist-bound structures display a hydrophobic moiety in this vicinity, forming hydrophobic contacts with residues from TMs 3, 5 and 6 (Fig. 3b).These groups also hinder the inward movement of the side chain of W319 6.48 through hydrophobic or π-π interactions (Fig. 3b).Mutagenesis data, such as L259 5.42 A, Y263 5.46 A or L322 6.51 A, further confirm the critical role of these hydrophobic contacts in sustaining antagonist efficacy (Supplementary Fig. S4f−h).
Regarding the selectivity differences, we observed that, in ET A , F161 3.28 rotates inwards to the orthosteric pocket in the antagonist-bound state, which results in a compact antagonistbinding pocket in ET A (Fig. 3c).In contrast, in the corresponding site in ET B , a smaller V177 3.28 yields a more expansive antagonistbinding pocket (Fig. 3d).This spatial variation accounts for the more effective accommodation of the larger bulky hydrophobic group (4-t-butylphenyl) of bosentan within ET B , while the smaller hydrophobic groups of macitentan and ambrisentan show a preference for ET A (Fig. 3e, f).Regarding zibotentan's ET A selectivity, as previously discussed, this may be attributed to the different residue Y129 2.53 in ET A compared to H150 2.53 in ET B (Figs. 2l, 3g).Together, Y129 2.53 and F161 3.28 in ET A may partially account for the observed selectivity of antagonists toward ET B .

Active and inactive conformation features of ETRs
To delve deeper into the activation mechanisms of ETRs, we determined the structures of ET A and ET B in their active states, at global resolutions of 3.3 Å for ET-1-bound ET A -miniG s/q -Nb35 complex and 3.2 Å for ET B -miniG s/q -Nb35 complex (Fig. 4a, b; Supplementary Fig. S6 and Table S1).In addition, a structure of ET B complexed with the selective agonist BQ3020 38 , was determined at a resolution of 3.0 Å (Fig. 4c; Supplementary Fig. S6a and Table S1).The overall conformations of ET-1-bound ET A -miniG s/q and ET B -miniG s/q complex structures, including the ET-1 binding poses, are consistent with previously reported ETR structures 30 (Supplementary Fig. S7a, b).Notably, the binding pose of BQ3020 in ET B closely resembles that of ET-1, with a C α RMSD of 0.8 Å for the receptor (Fig. 4d, f).BQ3020, which differs from the ET Bselective agonist IRL1620 by a single residue (Fig. 4e), exhibits an overall structural similarity, as the BQ3020-ET B and IRL1620-ET B complex structures display an RMSD of 0.9 Å.This indicates the aligned positioning of the agonists within the binding pocket (Fig. 4d).In the BQ3020-ET B complex, BQ3020's C-terminal configuration closely resembles that of ET-1, albeit with a slightly downward shift in the α-helix within the binding pocket, due to the absence of disulfide-bond constraints (Fig. 4d).
Additionally, the mutation W146 ECL1 A in ET A does not significantly affect receptor activation, whereas the analogous mutation W167 ECL1 A in ET B results in a substantial reduction in ET B activation (Supplementary Fig. S7c and Table S3).This suggests that ET B requires interactions with larger hydrophobic groups at this site for activation.Within ET B 's ECL1, F169 ECL1 is posited to engage in a π-π interaction with W167 ECL1 (Supplementary Fig. S7d), a key conformation for receptor function.Alanine mutation of F169 ECL1 hampers ET-1's capability to activate ET B (Supplementary Fig. S7c).
Employing the Residue-Residue Contact Score (RRCS) tool 39 , we analyzed structures bound by three antagonists, confirming that the residue contacts are characteristics of the inactive-state class A GPCRs (Supplementary Fig. S8).A comparative analysis of the macitentan-bound ET A structure against the ET-1-bound ET A structure reveals significant conformational changes when ET A transits from the inactive to active states.ET-1 binding promotes an inward movement of the extracellular portions of TM2, TM6 and TM7, resulting in a more compact receptor core (Fig. 5a).Concurrently, ECL2 moves inwards substantially, acting as a "lid" that secures ET-1 in place, facilitated by a π-π interaction between ET-1's Y13 and Y231 ECL2 in ET A (Fig. 5a).However, we cannot exclude the influence of Fab 301 on the conformational changes of ECL2 when compared to the state bound alone by the small-molecular antagonist.The EM density map allowed the modeling of extended N-terminal residues, revealing a tighter packing with ECL2 and ECL3 in the ET-1-bound ET A structure compared to the macitentan-bound state (Fig. 5a).These structural rearrangements lead to a more compact orthosteric pocket for ET-1 interaction.The characteristic outward displacement of the cytoplasmic part of TM6 by 9.7 Å in the ET-1-bound ET A -miniG s/q structure, reflects the conformational changes, indicative of class A GPCR activation (Fig. 5b).Moreover, the intracellular portion of TM7 undergoes a displacement of ~2.9 Å (measured by the C α atom of L369 7.53 ), in the active state (Fig. 5c).

Mechanism for ET A activation
The antagonist-bound ET A structures illuminate the underlying activation mechanism.Upon ET-1 binding, the side chain of I19 residue exerts pressure on I355 7.39 , prompting a downward shift of this residue and the associated intracellular half of TM7 (Fig. 5d).Concurrently, ET-1's W21 residue engages with the "toggle switch" residue W319 6.48 (Fig. 5e).This interaction induces a downward rotation of W319 6.48 , facilitating a hydrogen bond formation between its nitrogen atom and N361 7.45 (Fig. 5e).The downward motions of W319 6.48 and N361 7.45 promote an inward-to-outward rotation of F315 6.44 within the P 5.50 I/V 3.40 F 6.44 motif (Fig. 5e, f), triggering the outward movement of TM6 at the cytoplasmic end (Fig. 5e).These sequential events are accompanied by the disruption of interactions between L176 3.43 , L311 6.40 and V312 6.41 , which results in the release of the stacking of TM3 and TM6, further promoting the outward swing of TM6 (Fig. 5g).Additionally, the downward movement of N361 7.45 leads to the collapse of the Na + pocket, previously stabilized by D126 2.50 , T172 3.39 , and N361 7.45 in the inactive state, and triggers subsequent rearrangements between TM7, TM3 and TM2 (Fig. 5h).
Regarding the highly conserved N 7.49 P 7.50 xxY 7.53 motif in class A GPCRs, ET A and ET B feature a variant of N 7.49 P 7.50 xxL 7.53 Y 7.54 , wherein Y 7.53 is replaced by L 7.53 .During ET A activation, L179 3.46 does not engage in a polar inter-helix interaction, which permits L369 7.53 to move downward, enhancing its coupling with the of the G protein's α5 helix (Fig. 5c).Concerning the D 3.49 R 3.50 Y 3.51 motif, ET A activation disrupts the conserved ion lock between D182 3.49 and R183 3.50 .The released R183 3.50 then forms a polar interaction with Y275 5.58 and establishes interactions with the α5 helix of the G protein, anchoring the receptor in its active state (Fig. 5i).

Insights into antibody design to antagonize ET A
In our structural analysis of the antagonists-bound ET A , the employed Fab 301 shows antagonistic effect and facilitates the structure determination.The EM density maps clearly reveal the binding of Fab 301 to ET A 's ECL2 region (Fig. 6a).However, due to the dynamic nature of the interaction between Fab 301 and ET A 's ECL2, pinpointing their precise binding interface is challenging.We addressed this by using AlphaFold2 multimer 33,40 prediction to model the Fab 301 -ET A complex interface (Supplementary Fig. S9a).Ultimately the epitope of Fab 301 was roughly located based on the fitting of the predicted model with the experimental EM densities (Fig. 6c).
To pinpoint the exact ECL2 residues involved in binding, we performed an extensive alanine scanning mutagenesis.Our size exclusion chromatography analysis identified that the region spanning residues 230 ECL2 -235 ECL2 in ET A , and in particularly, residues R232 ECL2 and G233 ECL2 , are crucial for Fab 301 binding.Mutations of R232 ECL2 A or G233 ECL2 A resulted in a near-complete loss of Fab 301 's binding to ET A (Fig. 6b; Supplementary Fig. S10).These results complement our predicted binding mode, where R232 ECL2 of ET A forms an important polar interaction network with Fab 301 (Fig. 6c).Moreover, the hydrophobic interactions at this interface are crucial for antibody binding, as confirmed by our calcium mobilization assay on Fab 301 (Supplementary Fig. S9b).Within this model, E230 ECL2 establishes hydrogen bonds with the CDRL1 and CDRL2 regions of Fab 301 , whereas Y231 ECL2 and E234 ECL2 , together with R232 ECL2 , participate in a complex hydrogen bond network within the CDRH3 region of Fab 301 (Fig. 6c).Alanine mutations on E230 ECL2 , Y231 ECL2 , and R232 ECL2 also affected ET-1's activity on ET A .Notably, Y231 ECL2 mutation showed the most significant effect in calcium mobilization assay (Fig. 6d).These data underscore the essential role of ECL2 in the recognition of ligands and Fab 301 by ET A .
Comparative structural analysis of ET A bound by ET-1 and Fab 301 illustrates how ECL2 residues E230 ECL2 , Y231 ECL2 , and R232 ECL2 , which directly interact with ET-1, undergo a conformational change upon Fab 301 binding.This change fosters a polar interaction network with Fab 301 , effectively inhibiting ET-1 binding to ET A (Fig. 6f).Additionally, an ECL2 sequence alignment between ET A and ET B uncovers the basis for Fab 301 's selectivity for ET A over ET B (Fig. 6e).The distinct sequence variation, specifically at residues E230-Q235 of ET A , is pronounced between the two Fig. 4 Cryo-EM structures of BQ3020-ET B -miniG s/q -Nb35, ET1-ET B -miniG s/q -Nb35, and ET1-ET A -miniG s/q -Nb35 complexes.a-c Cryo-EM density maps of ET1-ET A -miniG s/q -Nb35 (a), ET1-ET B -miniG s/q -Nb35 (b), and BQ3020-ET B -miniG s/q -Nb35 (c) complexes.d Structural comparison of ET B bound to BQ3020, IRL1620, and ET-1.Conformational comparison between BQ3020 and IRL1620 in the binding pocket (upper right), and between BQ3020 and ET-1 (bottom right).The arrow indicates that the α-helix in the binding pocket shifts slightly downward.e Sequence alignment of peptides including ET-1, BQ3020 and IRL1620.f Cross-section of the BQ3020-binding pocket in ET B .BQ3020 is displayed as spheres (left panel).Detailed interactions between BQ3020 and ET B are shown (right panel).
receptors.This divergence provides a valuable template the design of selective antibodies targeting the ETR subtypes.

DISCUSSION
The structural elucidation of antagonist-bound ET A in this study provides critical insights into the mechanisms underpinning receptor selectivity and activity modulation.The cryo-EM structures of ET A in complex with macitentan, ambrisentan, and zibotentan underscore the intricate molecular interactions that govern antagonist binding.
The analysis of antagonist-bound ET A structures reveals conserved features aligning with the inactive state observed in antagonistbound ET B structures, suggesting common structural themes in ETR antagonism.Macitentan's high affinity to ET A is attributed to several key interactions, including hydrogen bonds and ionic interactions within the orthosteric pocket.The unique sulfonamide moiety enhances these interactions, resulting in a stable electrostatic network crucial for high binding affinity.Similarly, ambrisentan's high affinity and selectivity are due to its carboxylic acid group forming robust ionic and hydrogen bonds, coupled with hydrophobic interactions that create a snug fit within the binding pocket.Zibotentan's unique chair-like conformation and its interactions with residues around TM3, TM5, and TM6 further highlight the structural adaptations that facilitate selective binding.
The selectivity of antagonists for ET A over ET B is primarily influenced by specific residues within the binding pocket.F161 3.28 in ET A , which rotates inward in the antagonist-bound state, creates a compact binding pocket that favors smaller hydrophobic groups, whereas the corresponding V177 3.28 in ET B accommodates larger groups.The residue Y129 2.53 in ET A , compared to H150 2.53 in ET B , further contributes to this selectivity by engaging in critical polar interactions.
The antagonistic effect of Fab 301 and its ability to stabilize the ET A structure were utilized to gain further insights into antibody-ET A interactions.The binding of Fab 301 to the ECL2 region of ET A involves critical residues, notably R232 ECL2 and G233 ECL2 , essential for Fab 301 's binding affinity.The detailed mapping of the Fab 301 -ET A interface provides a template for designing selective antibodies that target specific ETR subtypes.
This study enhances our understanding of the structural basis for antagonist selectivity and activation mechanisms of ETRs.The high-resolution structures of antagonist-bound ET A reveal conserved features essential for selective binding and receptor stabilization.Key residues within the orthosteric pocket play crucial roles in determining antagonist affinity and selectivity, offering valuable insights into the design of selective therapeutic agents.

ET A and ET B construct design
For the complex structures of G protein-coupled ET A and ET B , the human ET A or ET B genes were cloned into pFastBac1 vector.This vector was modified to include a hemagglutinin (HA) signal peptide, a Flag tag at the N-terminus of the receptor, an HRV3C protease recognition site, and a 10× His tag at the C-terminus.To improve the protein yield of ET A , we fused an endoglucanase H (PDB: 2CIT) at the N-terminus of ET A , simultaneously truncating residues 1-49 at the N-terminus and residues 406-427 at the C-terminus.Similarly, for ET B , we truncated N-terminal residues 1-66 and C-terminal residues 407-442 and fused a sialidase H (PDB: 2VK5) to the N-terminus.

Expression and purification of Fab 301
As previously reported, Getagozumab 23 was used to produce the Fab 301 fragment.The Fab 301 fragment was codon-optimized and synthesized by GenScript.The corresponding light and heavy chain genes were then subcloned into the pFastBac Dual vector for expression using the Bac-to-Bac baculovirus expression system.Hi5 insect cells were infected with baculovirus at a density of 2 × 10 6 cells per mL and cultured at 27 °C.Cells were harvested 72 h post infection by centrifugation at 2000 rpm for 30 min, and the clear supernatant was collected.The pH of this supernatant was adjusted to 7.0 before the supernatant was applied to a 2 mL Ni-NTA resin and incubated at 4 °C for 2 h.The column was subsequently washed with a 15-CV buffer containing 20 mM HEPES, pH7.0, 500 mM NaCl, and 20 mM imidazole to remove nonspecifically bound proteins.The protein of interest was eluted from the column using an elution buffer containing 20 mM HEPES, pH 7.0, 100 mM NaCl, and 400 mM imidazole.The eluted protein fractions were then further purified on a Superdex 200 10/300 column, which was equilibrated in a buffer containing 20 mM HEPES, pH7.0, 100 mM NaCl, and 10% glycerol.Monomeric fractions were pooled, concentrated to 8.3 mg/mL, flash-frozen in liquid nitrogen, and stored at -80 °C for future use.
Fab 301 -ET A -anti-BRIL Fab-Nb complex formation and purification The purification of the ET A receptor was performed analogously to the method described above, with the addition of one of the antagonistsambrisentan, macitentan, or zibotentan during purification.The anti-Bril Fab was expressed in mammalian cells and purified following the protocol described previously 34 .The anti-Fab Nb was expressed in E. coli BL21(DE3) strain and purified according to the previously established methods 35 .
For complex formation, the ET A , Fab 301 , anti-BRIL Fab, and anti-Fab Nb were mixed at a molar mass ratio of 1:1.2:1.2:1.5.This mixture was incubated at 4 °C for 4 h, and then concentrated and applied to a Superdex 200 10/300 GL column preequilibrated with the buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.00075% (w/v) LMNG, 0.00015% (w/v) CHS, 0.00025% (w/v) GDN, 100 mM TCEP, and 50 μM of the chosen antagonist either ambrisentan, macitentan or zibotentan.Fractions containing the peak of interest were concentrated to an approximate concentration of 12 mg/mL for cryo-EM specimen preparation.

Cryo-EM sample preparation and data collection
A total of 3 μL of each complex sample, BQ3020-ET B -miniG s/q , ET1-ET B -miniG s/q , ET1-ET A -miniG s/q , Fab 301 -macitentan-ET A , Fab 301ambrisentan-ET A or Fab 301 -zibotentan-ET A , was applied to glowdischarged 300 mesh alloy grids (CryoMatrix Amorphous alloy film R1.2/ 1.3), and vitrified by Vitrobot Mark IV (Thermo Fisher Scientific).Excess sample was blotted by a filter paper for 3 s with a blot force of 2 before plunge-freezing in liquid ethane with a FEI Vitrobot Mark IV at 100% humidity and 4 °C.The frozen grids were transferred to liquid nitrogen and stored for data acquisition.Cryo-EM data collection was conducted with the Krios G4 cryo-transmission electron microscope (Thermo Fisher Scientific) operating at 300 kV, equipped with the Falcon 4 Direct Electron Detector (Thermo Fisher Scientific).Movies were recorded at a calibrated magnification of 130,000×, yielding a pixel size of 0.96 Å.A total dose of 60 electrons per square angstrom (e -/Å 2 ) was administered.Automated data collection was facilitated by the EPU software, utilizing a defocus range spanning from -1.0 μm to -2.0 μm.

Cryo-EM data processing
The overall cryo-EM data processing workflows for the BQ3020-ET B -miniG s/q , ET1-ET B -miniG s/q , ET1-ET A -miniG s/q , Fab 301macitentan-ET A , Fab 301 -ambrisentan-ET A and Fab 301 -zibotentan-ET A are shown in Supplementary Figs.S2 and S6.Cryo-EM movie stacks were corrected for beam-induced shifts utilizing the dose-weighting approach in Patch Motion Correction 43 .The contrast transfer function (CTF) parameters were calculated by employing the patch CTF estimation in CryoSPARC 44 .
For the BQ3020-ET B -miniG s/q -Nb35 complex, a total of 7685 images were imported in CryoSPARC v.4.0.1.A conventional neural network-based method Topaz 45 implemented in CryoSPARC, was used for particle picking.2,662,705 particles were extracted and then subjected to iterative 2D classification and ab initio reconstruction.Subsequently, 199,360 particles were selected for heterogeneous refinement.The best class was selected for homogeneous refinement, non-uniform refinement, and local refinement, generating a high-quality density map at a resolution of 3.0 Å. DeepEMhancer was applied to enhance local density.The processing steps for the ET1-ET B -miniG s/q -Nb35 and ET1-ET A -miniG s/q -Nb35 complexes, mirrored this approach, with specific details provided in Supplementary Fig. S6.
For Fab 301 -macitentan-ET A , Fab 301 -ambrisentan-ET A and Fab 301 -zibotentan-ET A complexes, the initial data processing steps were consistent with those of the G protein complexes.However, additional post-processing strategies were implemented.The Fab 301 -macitentan-ET A complex achieved high-quality density after the standard local refinement; thus, no further optimization was performed.In contrast, for the ambrisentan and zibotentan complexes, masks were generated post local refinement to omit the dynamic regions of Fab 301 .Detailed statistics on the number of images and particles at each processing stage are available in Supplementary Fig. S2.

For
Fab 301 -macitentan-ET A , Fab 301 -ambrisentan-ET A and Fab 301 -zibotentan-ET A complexes, the AlphaFold2-predicted ET A was used for the receptor modeling.The initial models for BRIL, anti-BRIL Fab and anti-Fab Nb were based on the structures derived from the GPR183 complex structure (PDB: 7TUY) 46 .For the BQ3020-ET B -miniG s/q -Nb35, ET1-ET B -miniG s/q -Nb35, and ET1-ET A -miniG s/q -Nb35 complexes, model building and refinement of the miniG s/q and Nb35 began with the miniG s/q structure from the GPR139-miniG s/q complex structure (PDB: 7VUH) 42 .Model docking into the EM density maps was carried out using Chimera 47 , followed by iterative manual adjustments and rebuilding in Coot 48 .Subsequent refinement was performed using phenix.real_space_refine in PHENIX 49 .Statistical validation of the model was conducted through MolProbity 50 .Structural figures were prepared using ChimeraX 51 .The complete refinement statistics are documented in Supplementary Table S1.

Intracellular calcium mobilization assay
CHO-K1 cells were cultured in Ham's F-12K (Kaighn's) Medium (Gibco-Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-Thermo Fisher Scientific), and 100 U/mL Penicillin-Streptomycin (Gibco-Thermo Fisher Scientific) in a humidified incubator at 37 °C with 5% CO 2 .Cells were seeded in 6-cm dishes overnight; when the density reached 60%-80%, the cells were transferred to F12K Medium supplemented with 10% FBS and transfected with 1.5 μg DNA encoding the ET A WT or mutants or 3 μg DNA encoding the ET B WT or mutants using TransIT2020 (Mirus Biosciences).The next day, transfected cells were harvested from the plate using Versene buffer (Gibco-Thermo Fisher Scientific) and seeded into black-sided, clear-bottomed 384-well plates (Agilent) at a density of 1,5000 cells per well.After 20 h, the 1% dFBS medium was removed, and cells were loaded with 20 μL/well of 1× Calcium 6 dye (Molecular Devices) and incubated at 37 °C for 1 h in the dark.10 μL/well of 3× ET-1 was added and the plates were read using the FLIPR Tetra High Throughput Cellular Screening System (Molecular Devices).To measure antagonist or Fab potency, 10 μL/well of 3× antagonist or Fab was added and incubated with the cells for 30 min at room temperature.The fluorescence intensity was recorded for 2 min after 10 μL/well of 4× ET-1 addition.All data were analyzed using GraphPad Prism 8 and the data are from least three independent replicate experiments.All plots are shown as means ± SEM.Data were determined using one-way ANOVA followed by Dunnett's multiple test compared with WT.The top value was normalized to 100% and the bottom value was normalized to 0% for the final presentation.Nonlinear curve fit was performed using a four-parameter logistic equation (log (agonist vs response) or log (inhibitor vs response)).

MD simulation
MD simulations were performed using the GROMACS2021 software, employing the CHARMM36m force field 52 and incorporating TIP3 water molecules.Parameters for ambrisentan were derived using the CGenFF force field 53 .To compensate for the missing ICL3 region in the ambrisentan complex structure, a segment from ET A in the G protein complex was used.The complete structure was prepared with the Protein Preparation Wizard in Maestro (2023-1, Schrödinger), which included the determination of the protonation states of residues at pH 7.4 using PROPKA.The protein was embedded into a lipid bilayer composing 150 POPC molecules using CHARMM-GUI 54 , which also facilitated the addition of 0.15 M sodium and chloride ions to neutralize the system's charge.Energy minimization and equilibration processes were performed following the default protocol of CHARMM-GUI, using a cutoff distance of 12 Å for nonbonded contacts and the Particle Mesh Ewald (PME) method 55 for long-range van der Waals interactions.Subsequent MD production runs of 500 ns were conducted at a temperature of 310 K and 1 bar using a semi-isotropic Parrinello-Rahman barostat.The final MD trajectories were analyzed and visualized using VMD 56 software, where the ligand RMSD calculations were also performed.

Fig. 1
Fig. 1 Cryo-EM structures of ET A in complex with macitentan, ambrisentan, zibotentan.a The inhibition activity of the four different antagonists on ET A in calcium mobilization assay.The IC 50 values of macitentan, ambrisentan, zibotentan and bosentan are 1.3 nM, 0.6 nM, 8.6 nM and 9.9 nM, respectively.Data are presented as means ± SEM (n = 5).b Cryo-EM density maps of Fab 301 -ET A -anti-BRIL Fab-Nb complex (left panel); zoomed-in view of the junction site and the surface presentation of Fab 301 (right panel).c-e Cartoon representation of Fab 301 -ET A -anti-BRIL Fab-Nb complexes with different antagonists: macitentan (c), ambrisentan (d), zibotentan (e).Components of ET A complexes are colored as indicated.The EM density map for each ligand is shown as colored mesh.

Fig. 3
Fig. 3 Structure basis of antagonist selectivity in ET A and ET B . a The sulfonylamide in macitentan and zibotentan, along with the carboxylic acid group in ambrisentan, engage with the positively charged area (blue surface) in ET A 's orthosteric pocket.b Three antagonists hinder the inward movement of W319 6.48 's side chain through analogous functional groups at the similar positions.c The conformation of F161 3.28 in different states.d In ET B 's corresponding position, V177 3.28 , offers a larger antagonist-binding pocket.e Different spatial accommodation of antagonists in ET A and ET B .The bulky hydrophobic group (4-t-butylphenyl) of bosentan is better adapted to ET B , whereas the smaller hydrophobic group of macitentan shows a preference for ET A .f Effects of V177 3.28 F in ET B on the antagonistic activities of ambrisentan and macitentan (n = 3).g Difference in pIC 50 values between ET A and ET B variants and WT in antagonist experiments.Data are presented as means ± SEM (n ≥ 3).All data were analyzed by one-way ANOVA by Dunnett's multiple test compared with WT.For mutants, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 were considered statistically significant.

Fig. 5
Fig. 5 Structural comparison of Fab 301 -macitentan-ET A -anti-BRIL Fab-Nb and ET1-ET A -miniG s/q -Nb35 complexes.The purple cartoon represents ET1-bound ET A , whereas cyan cartoon represents macitentan-bound ET A . a Conformational changes in loop regions and helical rearrangement during ET A activation (top view).b, c Structural comparison reveals the outward extension of TM6 (b) and the movement of the cytoplasmic portion of TM7 (c).d The downward movement of the side chain of I19 in ET-1-bound ET A structure.e-i Conformational changes of key motifs related to ET A activation.NPxxY (c), PIF motif (e, f), DRY (i).

Fig. 6
Fig. 6 Binding interface between Fab 301 and ET A . a Cryo-EM density maps showing the Fab 301 binding to the ECL2 region of ET A .b Differences between the retention time of each mutant-Fab 301 complex and the WT-Fab 301 complex.The letter "Δt" represents the retention time of the mutant sample minus the retention time of the WT sample.c The Fab 301 -ET A complex predicted by AlphaFold2 multimer was modeled, which fits well with the experimental electron density of Fab 301.Zoomed-in view of the antibody-binding interface is shown (right panel).d Response of ET-1 on WT ET A and ET A mutants in ECL2.Data are presented as means ± SEM (n = 4).All data were analyzed by one-way ANOVA by Dunnett's multiple test compared with WT. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 were considered statistically significant.e Sequence alignment of the ECL2 regions between ET A and ET B .Amino acids are classified according to their properties.f Conformational change of ECL2 between ET-1-bound ET A and Fab 301 -bound ET A .The gray area represents the Fab 301 density, and the arrow indicates that the ECL2 expands outward in the inactive structure.